Di-ester prodrugs of camptothecin, process for their preparation and their therapeutical applications

ABSTRACT

The present invention is related to 10,20-di-O ester derivatives of camptothecin and pharmaceutical formulations thereof. The compounds and pharmaceutical formulations of the present invention possess increased biological life span and bioavailability and reduced toxicity, while maintaining anti-cancer activity.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This patent application claims the benefit of U.S. Provisional PatentApplication No. 60/532,231, filed Dec. 23, 2003, which is incorporatedby reference herein.

FIELD OF THE INVENTION

The present invention relates the derivatives of camptothecin. Moreparticularly, the invention relates to di-ester derivatives ofcamptothecin, methods of preparing di-ester derivatives of camptothecinand pharmaceutical compositions comprising camptothecin di-esterderivatives.

BACKGROUND OF THE INVENTION

Camptothecin (CPT), shown in formula I:

was isolated and purified by Wall and coworkers in 1966 (J. Am. Chem.Soc. 88, 3888, (1966)). This compound was initially tested against themouse leukemia L 1210 system and found to be active. The compound wasquickly tested in human clinical trials. However, the subsequentbiological evaluation indicated that this compound is highly toxic andconsequently is unusable as a chemotherapeutic agent (Gottlieb et al.,Cancer Chemother. Rep. 54, 461, (1970), and 56, 103, (1972); Muggia etal., Cancer Chemother. Rep. 56, 515, (1972); Moertel et al., CancerChemother. Rep. 56, 95, (1972); and Schaeppi et al., Cancer Chemother.Rep. 5, 25, (1974)). The reason for the failure of the early trial waslater found to be due to the selection of an incorrect drug formulation.Camptothecin is insoluble in water. In order to use the drug forintravenous (iv) administration, camptothecin was converted to itssodium form (CPT sodium carboxylate). This form, although water-soluble,is practically devoid of anticancer activity. For example, a carefulevaluation of these agents in animal models made by Wani et al. revealedthat the sodium salt is only 10-20% as potent as the parent camptothecin(J. Med. Chem. 23, 554, (1980)). Important parameters for the anticanceractivity of camptothecin derivatives have now been established (Wall etal., Ann. Rev. Pharmacol. Toxicol. 17, 117, (1977)). The intact lactoneform with an α-hydroxyl group with the (S)-configuration at the C-20position of the molecule is essential for antitumor activity.Maintaining the molecule as an intact lactone is critical for successfultreatment.

Camptothecin and camptothecin derivatives are cytotoxic compounds whichcan be used as chemotherapeutic agents. The cytotoxic activity ofcamptothecin compounds is believed to arise from the ability of thesecompounds to inhibit both DNA and RNA synthesis and to cause reversiblefragmentation of DNA in mammalian cells. Camptothecin compounds inhibitthe enzyme DNA topoisomerase I which is known to relax supercoiled DNA.This relaxation is brought about by breakage of one of the DNA strandsin the formation of a covalent topoisomerase I-DNA complex. Camptothecinderivatives are believed to function by reversibly trapping theenzyme-DNA intermediate which is termed the “cleavable complex.” (Hsianget al. Cancer Research, 49, 4385, (1989)). The cleavable complex assaydeveloped by Hsiang et al. is a standard test for determining thecytotoxic activity of camptothecin compounds.

Camptothecin and its derivatives have shown a spectacular activityagainst a wide spectrum of human tumors grown in xenografts in nude mice(Giovanella et al., Cancer Res. 51, 3052, (1991), and Natelson et al.,Annals N.Y. Acad. Sci. 803, 224, (1996)), but much less activity wasobserved in human clinical trials. This difference in antitumor activityhas been associated with the finding that the hydrolysis of lactone tocarboxylate of the molecule is much faster in human plasma than inmouse. Burke and coworkers have systematically studied the stability ofcamptothecin derivatives in human serum (Annals N.Y. Acad. Sci. 803, 29,(1996)).

Ten-hydroxycamptothecin (10-HCPT) is a derivative of camptothecin, andalso a natural occurring compound. This compound was obtained as anaccompanying product on isolation of camptothecin and can now besynthesized from camptothecin in a number of ways. Currently, twoanti-cancer agents directly derived from 10-HCPT are commerciallyavailable for treatment. One is topotecan, and the other is irinotecan;and their structures are as follows:

The molecule 10-RCPT, is very potent against cancer cells.Unfortunately, this molecule is not useful for cancer treatment becauseof its toxicity. The molecule bears two hydroxyl groups, one each at theC-10 and C-20 positions. The C-20 hydroxyl group is adjacent to thecarbonyl group of the E-ring of the molecule, which constructs areactive α-hydroxy lactone moiety. This feature of the molecule makesthe lactone moiety very sensitive to hydrolysis, and thus, the moleculeis not stable in the body. The 10-phenolic hydroxyl group of 10-HCPT isnot stable in the process of enzymatic metabolism reactions. It is wellknown that phenolic hydroxy-containing moiety of an organic compound canbe enzymatically oxidized into a semi-quinone or quinone during theprocess of metabolism. The corresponding semi-quinone or quinonemetabolite is usually more toxic than the parental phenolic compound.

Thus, there is a need for protected 10-HCPT derivatives which are stablein the body and which have a longer biological life span.

Several reports have disclosed the esterification at the C-20 positionof camptothecin derivatives. U.S. Pat. Nos. 5,968,943 and 6,407,239,respectively, disclose the preparation of alkyl and aromatic esterproducts of camptothecins by introduction of an acyl group at the C-20position. U.S. Pat. Nos. 6,040,313 and 4,943,579 disclose theesterification of the hydroxyl group at the C-20 position ofcamptothecin compounds produces a non-toxic water-soluble prodrug. Theprodrug is non-toxic even though the parent camptothecin compound itselfmay be substantially more toxic. Hydrolysis of the ester formed at theC-20 position reforms the parent camptothecin compound afteradministration, thereby reducing the overall toxicity experienced by thepatient during camptothecin therapy.

U.S. Pat. No. 6,492,335 describes the glycoconjugates of camptothecinderivatives in which at least one carbohydrate component is linked viasuitable spacers with the 20-hydroxyl group of a camptothecinderivative. U.S. Pat. No. 6,376,617 reported the preparation of watersoluble polymeric conjugates of camptothecin withN-(2-hydroxypropyl)methacryloylamide linked via a spacer group to theC-20 position. The conjugates possess enhanced antitumor activity anddecreased toxicity with respect to the free drug. U.S. Pat. No.5,646,159 discloses the esterification of10,11-dioxymethylenecamptothecin with amino acid derivatives asacylating reagents at the C-20 position to provide several water-solublecompounds. U.S. Pat. No. 5,731,316 discloses the preparation of alkyl oralkenyl ester products of camptothecins by the esterification reactionat the C-20 position.

These patents disclose the single protection of the CPT molecule,meaning that the esterification reaction takes place at the C-20position. Although 10-HCPT and its derivatives are very potent againstcancer cells, they are not very useful due to high toxicities, lack ofstability, and shortened biological life spans. Thus, it is verydesirable to use the present invention which is able to overcome theproblems associated with prior art 10-HCPT and other camptothecinderivatives.

Microparticles and foreign bodies present in the blood are generallycleared from the circulation by the “blood filtering organs”, namely thespleen, lungs and liver. The particulate matter contained in normalwhole blood comprises red blood cells (typically 8 microns in diameter),white blood cells (typically 6-8 microns in diameter), and platelets(typically 1-3 microns in diameter). The microcirculation in most organsand tissues allows the free passage of these blood cells. Whenmicrothrombii (blood clots), with the size greater than 10-15 microns,are present in circulation, a risk of infarction or blockage of thecapillaries will be generated, leading to ischemia or oxygen deprivationand possible tissue death. Injection into the circulation of particlesgreater than 10-15 microns in diameter, therefore, must be avoided. Asuspension of particles less than 7-8 microns, is however, relativelysafe and has been used for the delivery of pharmacologically activeagents in the form of liposomes and emulsions, nutritional agents, andcontrast media for imaging applications.

The size of particles and their mode of delivery determine theirbiological behavior. Strand et al. (in Microspheres-BiomedicalApplications, ed. A. Rembaum, pp 193-227, CRC Press (1988)) havedescribed the fate of particles to be dependent on their size. Particlesin the size range of a few nanometers (nm) to 100 nm enter the lymphaticcapillaries following interstitial injection, and phagocytosis may occurwithin the lymph nodes. After intravenous/intraarterial injection,particles less than about 2 microns will be rapidly cleared from theblood stream by the reticuloendothelial system (RES), also known as themononuclear phagocyte system (MPS). Particles larger than about 7microns will, after intravenous injection, be trapped in the lungcapillaries. After intraarterial injection, particles are trapped in thefirst capillary bed reached. Inhaled particles are trapped by thealveolar macrophages.

Intravenous drug delivery permits rapid and direct equilibration withthe blood stream which carries the medication to the rest of the body.To avoid the peak serum levels which are achieved within a short timeafter intravascular injection, administration of drugs carried withinstable carriers would allow gradual release of the drugs inside theintravascular compartment following a bolus intravenous injection of thetherapeutic nanoparticles.

Injectable controlled-release nanoparticles can provide a pre-programmedduration of action, ranging from days to weeks to months from a singleinjection. They also can offer several profound advantages overconventionally administered medicaments, including automatic assuredpatient compliance with the dose regimen, as well as drug targeting tospecific tissues or organs (Tice and Gilley, Journal of ControlledRelease 2, 343-352 (1985)).

Pharmaceuticals that are water-insoluble or poorly water-soluble andsensitive to acid environments in the stomach cannot be conventionallyadministered (e.g., by intravenous injection or oral administration).The parenteral administration of such pharmaceuticals has been achievedby emulsification of the oil solubilized drug with an aqueous liquid(such as normal saline) in the presence of surfactants or emulsionstabilizers to produce stable microemulsions. These emulsions may beinjected intravenously, provided the components of the emulsion arepharmacologically inert. U.S. Pat. No. 4,073,943 describes theadministration of water-insoluble pharmacologically active agentsdissolved in oils and emulsified with water in the presence ofsurfactants such as egg phosphatides, pluronics (copolymers ofpolypropylene glycol and polyethylene glycol), polyglycerol oleate, etc.PCT International Publication No. WO85/00011 describes pharmaceuticalmicrodroplets of an anaesthetic coated with a phospholipid such asdimyristoyl phosphatidylcholine having suitable dimensions forintradermal or intravenous injection.

SUMMARY OF THE INVENTION

The present invention provides novel 10,20-di-ester products of 10-HCPT.In keeping with the present invention, both the C-10 and C-20 positionsof 10-HCPT are protected by the introduction of acyl groups into the10-HCPT, one each at the C-10 and C-20 positions. The compounds of theinvention significantly increase the biological life span andbioavailability of 10-HCPT while maintaining the inherent anti-canceractivity of 10-HCPT and lowering the toxicity of 10-HCPT.

According to the present inventions, 10,20-di-ester of 10-HCPTderivatives can be activated in vivo and the parent drug is protected.Conversion of the prodrugs to camptothecin is mediated by a group ofenzymes called esterases that are present in the blood of many animals,including humans. Since the prodrugs are rapidly distributed throughoutthe body within a short period of time after delivery, these prodrugsexist at a very low concentration at the time they undergo enzymatichydrolysis. This prevents camptothecin from precipitating in the bloodstream.

By means of the ester-like linkage of the carrier radicals to the C-20hydroxyl group, the lactone ring in the camptothecin derivatives, whichis important for the action, is stabilized. Compared with the underlyingtoxophores, they have markedly higher tolerability and tumor selectivityand improved solubility.

The introduction of ester functionality at the C-10 position of CPTderivatives will slowly release the active form of 10-hydroxy CPT, whichworks as the anticancer reagent.

Thus, the compounds disclosed in this invention significantly increasethe biological life span and bioavailability of camptothecins whilemaintaining the inherent anti-cancer activity and lowering the toxicityof the camptothecins.

The present invention also provides methods to enhance drugbioavailability of the di-ester camptothecin derivatives. The presentinvention further provides a new method, nanoparticle technology, forthe formulation and delivery of such hydrophobic products.

The nanoparticle formulation of this present invention provides newdelivery system for the camptothecin derives.

Accordingly, the present invention provides new 10-HCPT analogs, whichare active against various different types of tumors. The presentinvention also provides 10,20-di-O-ester products of 10-HCPT, and which,in some embodiments, are water-soluble derivatives thereof.

The present invention further provides prodrugs of 10-HCPT derivatives.The prodrugs can release the parent active 10-HCPT compounds, believedto be by an enzymatic cleavage of 10,20-di-O-ester, after reaching thetargeting organs.

The present invention also provides an improved treatment for certaintypes of cancers using the di-ester camptothecin derivatives describedherein.

Additional objects and advantages of the present invention will be setforth in the detailed description of the preferred embodiments whichfollows.

DETAILED DESCRIPTION OF THE INVENTION

Conversion of the compounds disclosed by the present invention to theparental 10-HCPT derivatives is mediated by a group of enzymes calledesterases. Mammalian carboxylesterases represent a multigene family andare present in a wide variety of organs and tissues of many mammalianspecies (Satoh, in reviews in Biochemical Toxicology, 8:155-81, NewYork: Elsevier, (1987); Heymann, in Enzymatic Basis of Detoxication,2:291-323, New York: Academic, (1980), and in Metabolic Basis ofDetoxication, 1:229-45, New York: Academic, (1982)). More informationabout distribution of carboxylesterases in tissues can be found in areview article written by Satoh et al. (Annu. Rev. Pharmacol. Toxicol.38, 257, (1998)). Carboxylesterases are known to be responsible for thehydrolysis of many exogenous compounds, the consequences of whichinclude both activation of prodrugs and deactivation of drugs.Irinotecan, as discussed above, is a prodrug of 7-ethyl-10-HCPT (SN-38).This compound is converted to SN-38 by carboxylesterases (Danks et al.,Cancer Res. 58, 20, (1998); Potter et al., Cancer Res. 58, 2646, (1998);Tsuji et al., J. Pharmacobio-Dyn. 14, 341, (1991)). The compounds havinga formula II as disclosed by the present invention are rapidlydistributed throughout the body within a short period of time afteradministration, and the di-esters at the positions of C-10 and C-20(respectively) are subsequently cleaved to release the active parentalcompounds by carboxylesterases specifically in organ tissues.

The di-ester compounds of the present invention are active in inhibitingtopoisomerase I and yet are non-toxic over a wide active dose range.Those compounds enable one to administer greater amounts of the activecamptothecin compound as its non-toxic ester prodrug while avoiding thetoxicity of the parent compound. Slow hydrolysis of the ester groups atthe C-10 and C-20 positions to yield the free hydroxyl group, results inthe slow controlled formation of the parent compound afteradministration of the di-ester prodrug. The slow formation of the parentcompound is less toxic than administration of the corresponding amountof the parent compound initially. That is, the present invention allowsone to administer a much larger dose of camptothecin compound as theprodrug than as the corresponding parent camptothecin compound. Forexample, the present invention allows one to administer a 10 foldgreater amount of the ester (which is hydrolyzed to the parent compound)than the parent compound itself. While not being bound to any particulartheory, it is believed that the di-ester prodrug is slowly hydrolyzed tothe parent camptothecin compound limiting damage to cellular tissues, inparticular blood cells. The non-toxicity of the compounds of the presentinvention is an important improvement over prior art camptothecincompounds.

In accordance with the present invention, there are provided 10-HCPTdi-ester derivatives with the general formula II:

wherein

R is the same or different and R is C₁-C₃₀ alkyl, C₂-C₂₂ alkenyl, C₄-C₂₀aryl, (CH₂)_(n)OR₅, (CH₂)_(n)SR₅, (CH₂)_(n)NR₅R₆, (CH₂)_(n)COR₇.

R₁, R₂, R₃, and R₄ are the same or different, and are hydrogen, halo,C₁-C₂₀ alkyl, C₁-C₈ alkoxyl, C₄-C₂₀ aryl, and C₁-C₂₀ silyl.

n is an integer of 1 to 8,

R₅ and R₆, which can be the same or different, are C₁-C₈ alkyl, C₂-C₆alkenyl and C₄-C₁₀ aryl,

R₇ is hydroxy, C₁-C₆ alkoxy, C₂-C₆ alkenyl, C₄-C₂₀ aryl or NR₈R₉,

wherein R₈-R₉, which can be the same or different, are C₁-C₆ alkyl.

An illustrative embodiment of the present invention is a compound offormula II, wherein

R is the same or different and R is C₁-C₂₀ alkyl, C₂-C₆ alkenyl, C₄-C₂₀aryl, (CH₂)_(n)OR₅, (CH₂)_(n)SR₅, (CH₂)_(n)NR₅R₆, (CH₂)_(n)COR₇.

R₁, R₂, R₃, and R₄ are the same or different, and are hydrogen, halo,C₁-C₂₀ alkyl, C₁-C₈ alkoxyl, C₄-C₂₀ aryl, and C₁-C₂₀ silyl.

n is an integer of 1 to 8,

R₅ and R₆, which can be the same or different, are C₁-C₈ alkyl, C₂-C₆alkenyl and C₄-C₁₀ aryl,

R₇ is hydroxy, C₁-C₆ alkoxy, C₂-C₆ alkenyl, C₄-C₂₀ aryl or NR₈R₉,

wherein R₈-R₉, which can be the same or different, are C₁-C₆ alkyl.

The invention also provides pharmaceutically acceptable salts of the10-HCPT-di-ester derivatives described above.

The following definitions refer to the various terms used above andthroughout the disclosure.

The term “halo” refers to fluoro, chloro, bromo or iodo.

The term “C₁-C₃₀ alkyl” refers to an alkyl, substituted straight orbranched chain alkyl or alkylenyl group, having from 1-30 carbon atoms.In view of availability of alkylating reactants, the alkyl group haspreferably 1-22 carbon atoms. Illustrative of the alkyl group includethe methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl,t-butyl, pentyl, 3-methylbutyl, 2,2-dimethylpropyl, 1,1-dimethylpropyl,hexyl, 1-methylpentyl, 4-methylpentyl, heptyl, 1-methylhexyl,2-methylhexyl, 5-methylhexyl, 3-ethylpentyl, octyl, 2-methylheptyl,6-methylheptyl, 2-ethylhexyl, 2-ethyl-3-methylpentyl,3-ethyl-2-methylpentyl, nonyl, 2-methyloctyl, 7-methyloctyl,4-ethylheptyl, 3-ethyl-2-methylhexyl, 2-ethyl-1-methylhexyl, decyl,2-methylnonyl, 8-methylnonyl, 5-ethyloctyl, 3-ethyl-2-methylheptyl,3,3-diethylhexyl, undecyl, 2-methyldecyl, 9-methyldecyl, 4-ethylnonyl,3,5-dimethylnonyl, 3-propyloctyl, 5-ethyl-4-methyloctyl, 1-pentylhexyl,dodecyl, 1-methylundecyl, 10-methylundecyl, 3-ethyldecyl, 5-propylnonyl,3,5-diethyloctyl, tridecyl, 11-methyldodecyl, 7-ethylundecyl,4-propyldecyl, 5-ethyl-3-methyldecyl, 3-pentyloctyl, tetradecyl,12-methyltridecyl, 8-ethyldodecyl, 6-propylundecyl, 4-butyldecyl,2-pentylnonyl, pentadecyl, 13-methyltetradecyl, 10-ethyltridecyl,7-propyldodecyl, 5-ethyl-3-methyldodecyl, 4-pentyldecyl, 1-hexylnonyl,hexadecyl, 14-methylpentadecyl, 6-ethyltetradecyl, 4-propyltridecyl,2-butyldodecyl, heptadecyl, 15-methylhexadecyl, 7-ethylpentadecyl,3-propyltetradecyl, 5-pentyldodecyl, octadecyl, 16-methylheptadecyl,5-propylpentadecyl, nonadecyl, 17-methyloctadecyl, 4-ethylheptadecyl,icosyl, 18-methylnonadecyl, 3-ethyloctadecyl, henicosyl, docosinyl,tricosinyl, tetracosinyl and pentacosinyl groups.

The term “C₂-C₂₂ alkenyl” represents an alkenyl group, this has from 2to 22 carbon atoms, and may be a straight or branched chain group,preferably, natural or unnatural fatty acid. It may have 1 or more,preferably from 2 to 6, double bonds. Examples of such groups includethe vinyl, allyl, 1-propenyl, isopropenyl, 2-methyl-1-propenyl,1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl,4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl,1-heptenyl, 2-heptenyl, 3-heptenyl, 1-octenyl, 8-nonenyl, 1-nonenyl,1-decenyl, 9-decenyl, 8-tridecenyl, cis-8-pentadecenyl,trans-8-pentadecenyl, 8-heptadecenyl, 8-heptadecenyl,8,11-heptadecadienyl, 8,11,14-heptadecatrienyl,4,7,11,14-nonadecatetraenyl and2,6-dimethyl-8-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1,3,5,7-nonatetraen-1-yl,cis-10-nonadecaenyl, 10,13-nonadecadienyl, cis-7,10,13-nonadecatrienyl,5,8,11,14-nonadecatetraenyl, nonadedapentaenyl, henecosatetraenyl,henecosapentaenyl, henecosahexaenyl, myristyl, and eicosyl groups.

When the alkyl groups are branched, the branched chains may form acycloalkyl group such as cyclopentyl, cylohexyl, cylcoheptyl, orcyclooctyl group.

The term “C₁-C₈ alkoxy” refers to an alkoxy group with one to eightcarbon alkyl groups, and the alkyl moiety thereof generally correspondsto the C₁-C₃₀ alkyl groups describes above and can be selectedtherefrom. Examples of alkoxy groupsare those derived from straight orbranched chain lower alkyl groups with 1-8 carbon atoms, and include,for example, methoxy, ethoxy n-propoxy, isopropoxy, n-butoxy, isobutoxy,sec-butoxy, tert-butoxy, n-pentyloxy, isopentyloxy, n-hexyloxy,cyclohexoxy, n-heptyloxy, n-octyloxy and 2-ethylhexyloxy.

The term “C₄-C₂₀ aryl” refers to an aromatic or heteroaromatic ring,including by way of example, phenyl, naphthyl, furanyl imidazolyl andthionyl. The aryl ring can be substituted with substituents selectedfrom the group consisting of halo, C₁-C₆ alkyl or C₁-C₆ alkoxy, or alkylamino. Examples include 4-chlorophenyl, 2-fluorophenyl, 4-fluorophenyl,3-fluorophenyl, 4-methylphenyl, 4-ditrifluorohenyl, 2-ethylphenyl,3-n-propylphenyl, 4-isopropyl-phenyl, 4-n-butylphenyl, 4-t-butylphenyl,4-sec-butylphenyl, 4-dimethylaminophenyl, 3,4-dimethylphenyl,4-methoxyphenyl, 4-ethoxyphenyl, 4-isopropoxyphenyl, 3-isobutoxyphenyl,4-t-butoxyphenyl, 4-nitrophenyl, 2-furan, 2-pyridyl, 3-pyridyl,2-thiophenyl, 3-thiophenyl, 1-naphthyl, 2-naphthyl, 2-indolyl and thelike.

The term “(CH₂)_(n)OR₅, (CH₂)_(n)SR₅ and (CH₂)_(n)NR₅R₆” refers to thealkyl groups substituted with oxygen, sulfur and nitrogen, wherein R₅and R₆ include hydrogen, or C₁-C₆ alkyl groups, preferably C₁-C₄ alkylgroups, C₂-C₆ alkenyl groups, C₄-C₁₀ aryl groups, and n is an integer of1 to 8, preferably, n is the integer 1, 2 or 3. Preferred examples ofthe alkyl group substituted with oxygen, sulfur or nitrogen includemethoxymethyl, ethoxymethyl, propoxymethyl, n-butoxymethyl,2-methoxyethyl, 2-ethoxyethyl, 2-propoxyethyl, 3-methoxypropyl,3-ethoxypropyl, 3-propoxypropyl, 4-methoxybutyl, 4-propoxybutyl,dimethoxymethyl, 2,2-dimethoxyethyl, diethoxymethyl, 2,2-diethoxyethyl,dipropoxymethyl and 2,2-dipropoxyethyl groups. Preferred examples of(CH₂)_(n)SR₅ are methylthiomethyl, ethylthiomethyl, propylthiomethyl,n-butylthiomethyl, 2-methylthiolethyl, 2-ethylthiolethyl,2-propylthiolethyl, 3-methylthiopropyl, 3-ethylthiopropyl,3-propylthiopropyl, 4-methylthiobutyl, and 4-propylthiobutyl groups.Preferred examples of (CH₂)_(n)NR₅R₆ are aminomethyl,dimethylaminomethyl, (N-acetyl)methylaminomethyl, diethylaminomethyl,dipropylaminomethyl, dibutylaminomethyl; dimethylaminoethyl,diethylaminoethyl, dipropylaminoethyl, and dibutylaminoethyl groups.

The term “(CH₂)_(n)COR₇” refers to carboxylic acid, ester, or amide,wherein n is an integer of 1 to 8, preferably n is an integer 1, 2, 3 or4, and R₇ is hydroxy, C₁-C₆ alkoxy or NR₈R₉. When R₇ is NR₈R₉, R₈ and R₉can be the same or different and are alkyl or substituted alkyl groupswith one to six carbon atoms. The alkyl moiety of R₈ and/or R₉ generallycorrespond to the C₁-C₂₀ alkyl groups discussed above and can beselected therefrom. Preferred examples of the alkylamino group are thosederived from hydrogen, and straight or branched chain lower alkyl groupswith 1-6 carbon atoms, such as methyl, ethyl, propyl, butyl, pentyl andhexyl groups.

The term “C₃-C₂₀ silyl” refers to an Si(R₁₀R₁₁R₁₂), wherein R₁₀, R₁₁,R₁₂ can be the same or different and are the substituted straight orbranched chain alkyl or alkenyl groups and generally has 3-20 carbonatoms. The alkyl moiety thereof generally corresponds to the aforesaidalkyl group. Preferred illustrative examples are trimethylsilyl,triethylsilyl, tributylsilyl, t-butyldimethylsilyl, t-butyldiethylsilyland t-butyldiphenylsilyl.

In the above general formula (II), either one of the substituents R₁ andR₂ is preferably hydrogen. The radical R₃ located in the C-9 position ispreferably a halogen atom, nitro or an alkyl group of 1-10 carbon atoms,with a substitution, such as chloro, amino, or alkylamino groups beingpreferred. The alkyl moiety is preferably a lower alkyl or substitutedalkyl group with 1-8 carbon atoms. The alkyl moiety thereof generallycorresponds to the C₁-C₂₀ alkyl group discussed above. Illustrative ofthe substituted alkyl groups are, for example, aminomethyl, aminoethyl,aminopropyl, dimethylaminomethyl, dimethylaminoethyl,dimethylaminopropyl, diethylaminomethy, diethylaminoethy,diethylaminopropyl. The alkylamino groups are, for example, methylamino,ethylamino, propylamino, isopropylamino, n-butylamino, tert-butylamino,pentylamino, hexylamino, heptylamino and octylamino groups.

In the above general formula (II), R₄ located in the C-7 positionrepresents halo, phenyl, an alkyl group or a Si(R₁₀R₁₁R₁₂), group.Preferably, R₄ is hydrogen, a halogen atom or an alkylsilyl, with thealkylsilyl group being most preferable, such as trimethylsilyl,t-butyldimethylsilyl, triethylsilyl, and t-butyldiphenylsilyl. In caseof R₄ being a substituted or unsubstituted phenyl group, preferableexamples of the phenyl-substituted alkyl group include phenoxymethyl,p-methylphenoxymethyl, o-chlorophenoxymethyl, 2-phenoxyethyl,3-phenoxypropyl and 4-phenoxybutyl group.

In the above general formula (II), R is preferably an unsubstituted orsubstituted alkyl group, aromatic group, or heteroaromatic group. Themost preferable substituents on the aromatic or heteroaromatic ring arehydrogen, halogen, C₁-C₄ alkyl, NR₈R₉ and the like. More preferably, thesubstituted alkyl groups are (CH₂)_(n)OR₅, (CH₂)_(n)SR₅, (CH₂)_(n)NR₅R₆wherein n is either 1 or 2, and (CH₂)_(n)COR₇ wherein n is 1, 2, 3 or 4,and R₅ to R₉, which can be the same or different, are the same asdescribed above. Illustrative examples of the preferred R groups areaminomethyl, dimethylaminomethyl, methoxymethyl, methoxyethyl,methoxyethyl, ethyl, propyl, hexyl, phenyl, 4-chlorophenyl,2-fluorophenyl, 4-fluorophenyl, 3-fluorophenyl, 4-methylphenyl,4-ditrifluorohenyl, 3,4-dimethylphenyl, 4-methoxyphenyl, 4-ethoxyphenyl,4-isopropoxyphenyl, 4-nitrophenyl, 2-furan, 3-pyridyl, 2-thiophenyl andthe like. The most preferable di-esters of 10-HCPT in accordance withthe invention are methoxylacetate, ethoxylacetate, propoxylacetate,glycinate, methylaminoacetate, dimethylaminoacetate,diethylaminoacetate, benzoate, fluorobenzoate, methoxy benzoate,succinate, and glutate.

According to the present invention, any camptothecin compounds havingavailable hydroxyl groups at the C-10 and C-20 positions may be used toprepare the di-ester derivatives. Suitable camptothecin compounds aredescribed, for example, in U.S. Pat. No. 4,545,880, U.S. Pat. No.4,604,463, and U.S. Pat. No. 4,473,692. These patents are incorporatedherein by reference for a more complete description of camptothecincompounds which can be used to prepare the di-esters of the presentinvention.

Camptothecin compounds having the following general structure asdepicted by the general formula III:

can be converted to the di-ester camptothecin derivatives of the presentinvention.

In the camptothecin compounds of formula III, the substituents R₁, R₂,R₃, and R₄ are not critical and can be any of a wide variety of groupsand combinations of groups, such as H, and unsubstituted and substitutedalkyl, alkenyl, alkoxy, silyl, aryl, and alkylsilyl groups, and thelike. Preferably, R₁, R₂, R₃, and R₄ generally correspond to R₁, R₂, R₃,and R₄ of the compounds of the invention, formula II, as describedabove.

Illustrative examples of the preferred 10-HCPT derivatives that areuseful starting materials to form the compounds of the present inventionare, 10-hydroxycamptothecin (10-hydroxy-CPT; “camptothecin” is referredto hereinafter simply as “CPT”), 10-hydroxy-7-methyl-CPT,10-hydroxy-7-ethyl-CPT, 10-hydroxy-7-propyl-CPT,10-hydroxy-7-benzyl-CPT, 10-hydroxy-7-trimethylsilyl-CPT,10-hydroxy-7-triethylsilyl-CPT, 10-hydroxy-7-dimethylbutylsilyl-CPT,10-hydroxy-7-triethylsilyl-CPT, 10-hydroxy-9-nitro-CPT,10-hydroxy-9-dimethylamino-CPT, 10-hydroxy-9-diethylamino-CPT. The mostpreferable analogs, as starting materials for the present invention, are10-HCPT, 7-ethyl-10-CPT and 7-tert-butyldimethylsilyl-10-HCPT and9-dimethylamino-10-HCPT.

In accordance with the present invention, there is also provided theprocesses for the preparation of various 10-HCPT di-ester derivatives ofthe general formula (II).

The compounds of the present invention are prepared by esterifying the10,20-position hydroxyl groups of a camptothecin compound to generatethe desired product.

Generally, the reaction of 10-HCPT derivatives with the correspondingacylating reagents forms the di-ester products. The preparation ofcompounds of formula (II) follows the general synthetic procedures thatare known in the literature.

The di-ester can be prepared from the corresponding anhydride in thepresence of a catalytic amount of acid at elevated temperature. Forexample, 10-HCPT, 3 to 50 molar equivalent of the acylating reagent withthe general formula R′CO—O—COR′, and a catalytic amount of concentratedH₂SO₄ was added to a round-bottomed flask equipped with a magneticstirrer. The mixture was stirred at elevated temperature (100±10° C.)under nitrogen gas for 12-48 hours. After cooling to room temperature,the mixture was poured into petroleum ether portion by portion whilestirring, and the precipitates were collected by filtration and thendissolved into dichloromethane/water. The separated organic layer waswashed with 5% NaHCO₃, brine, dried over sodium sulfate, andconcentrated. The residue was purified by flash silica gelchromatography eluted with tetrahydrofuran/dichloromethane to afford thedesired product in the yield of 10 to 90%.

In this way, any anhydrides can be used for the invention to obtain thedesired di-ester product, such as aliphatic, aromatic carboxylic, andheterocyclic carboxylic anhydride. Generally, the anhydride can beprepared from the corresponding acid with the general synthetic methodin the prior art.

The di-ester prodrugs of the present invention are also prepared byesterifying the 10,20-hydroxyl groups of camptothecins with thecorresponding alkyl or aromatic carboxylic acids. The reaction can becarried out in anhydrous solvent (such as dimethylformamide and methylsulfoxide) in the presence of one equivalent of dicyclohexylcarbodiimide(DCC) and a catalytic amount of an amine base, preferably a secondary ortertiary amine. Any precipitate which form is removed by filtration andthe product is isolated after removal of the solvent. The desiredproduct can be purified with column chromatography. Thus, for example,7-ethyl-10-HCPT may be allowed to react in methyl sulfoxide with a molarexcess, for example up to five-fold molar excess or more, especially 2mol equivalents, of an acid in anhydrous dimethylformamide in thepresence of DCC and 4-dimethylaminopyridine, to afford the desireddi-ester product.

Any acid can be used for the invention to obtain the desired di-esterproduct, including, for example, aliphatic acids, aromatic carboxylicacids, heterocyclic carboxylic acids, and aralkylcarboxylic acids. Theseacids may contain one or more unsaturated bonds in the molecule andcarry one or more substituents such as halogen atoms, amino groups andhydroxyl groups, such as various kinds of amino acids. The illustrativeexamples are chloroacetic acid, propionic acid, butyric acid,phenylacetic acid, succinic acid, glutaric acid, adipic acid, glycine,alanine, valine, leucine, isoleucine, phenylalanine, tyrosine,tryptophan, leucine, arginine, histidine, aspartate, glutamate,asparagine, glutamine, cysteine, methionine and their derivatives.

The amino or alkylamino groups of di-esters can be converted to an acidaddition salt by the addition of a pharmaceutically acceptable acid.Suitable acids include both inorganic and organic acids. Suitableaddition salts include, but are not limited to hydrochloride, sulfate,phosphate, diphosphate, hydrobromide, nitrate, acetate, malate, maleate,fumarate, tartrate, succinate, citrate, lactate, methanesulfonate,p-toluenesulfonate, palmoate, salicylate and stearate salts. The saltscan be purified by crystallization from a suitable solvent, or dissolvedin water and lyophilized.

The compounds disclosed in the present invention are prodrugs of 10-RCPTderivatives. 10-HCPT di-ester derivatives are very active againstcancers, but very toxic as well. The compounds of the present inventiondo not only exhibit the anti-cancer activity of the 10-HCPT derivatives,but they also greatly decrease the toxicity of their parental compound.Thus, the compounds of the present invention can be very effective inthe treatment of cancers, including, but not limited to, human cancersof the lung, breast, colon, prostate, melanoma, pancreas, stomach,liver, brain, kidney, uterus, cervix, ovaries urinary track,gastrointestinal, and other solid tumors which grow in an anatomicalsite. Other solid tumors include, but not limited to, colon and rectalcancers. The compounds of the present invention are also effective inthe treatment of the other types of tumors growing in the blood streamand blood borne such as leukemia.

The di-ester of 10-HCPT of the present invention can be administered asa pharmaceutical composition containing the compounds and apharmaceutically acceptable carrier or diluent. The active materials canalso be mixed with other active materials which do not impair thedesired action and/or supplement the desired action. The activematerials according to the present invention can be administered by anyacceptable route including, but not limited to, orally, parenterally,intravenously, intradermally, subcutaneously, through an inhaler ortopically, in liquid or solid form.

Oral compositions will generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, theaforesaid compounds can be incorporated with excipients and used in theform of tablets, troches, capsules, elixirs, suspensions, syrups,wafers, chewing gums and the like.

The tablets, pills, capsules and the like can contain the followingingredients: a binder such as microcrystalline cellulose, gum tragacanthor gelatin; an excipient such as starch or lactose, a disintegratingagent such as alginic acid, corn starch and the like; a lubricant suchas magnesium stearate; a glidant such as colloidal silicon dioxide; anda sweetening agent such as sucrose or saccharin or flavoring agent suchas peppermint, methyl salicylate, or orange flavoring may be added. Whenthe dosage unit form is a capsule, it can contain, in addition tomaterial of the above type, a liquid carrier such as a fatty oil. Otherdosage unit forms may contain other various materials which modify thephysical form of the dosage unit, for example, as coatings. Thus,tablets or pills can be coated with sugar, shellac, or other entericcoating agents. A syrup can contain, in addition to the activecompounds, sucrose as a sweetening agent and certain preservatives, dyesand colorings and flavors. Materials used in preparing these variouscompositions should be pharmaceutically or veterinarally pure andnon-toxic in the amounts used.

For the purposes of parenteral therapeutic administration, the activeingredient can be incorporated into a solution or suspension. Thesolutions or suspensions can also include the following components: asterile diluent such as water for injection, saline solution, fixedoils, polyethylene glycols, glycerine, propylene glycol or othersynthetic solvents; antibacterial agents such as benzyl alcohol ormethyl parabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. The parenteral preparationcan be enclosed in ampoules, disposable syringes or multiple dose vialsmade of glass or plastic.

The pharmaceutical forms suitable for injectable use include sterilesolutions, dispersions, emulsions, and sterile powders. The final formmust be stable under conditions of manufacture and storage. Furthermore,the final pharmaceutical form must be protected against contaminationand must, therefore, be able to inhibit the growth of microorganismssuch as bacteria or fungi. A single intravenous or intraperitoneal dosecan be administered. Alternatively, a slow long-term infusion ormultiple short-term daily infusions may be utilized, typically lastingfrom 1 to 8 days. Alternate day or dosing once every several days mayalso be utilized.

Sterile, injectable solutions are prepared by incorporating a compoundin the required amount into one or more appropriate solvents to whichother ingredients, listed above or known to those skilled in the art,may be added as required. Sterile injectable solutions are prepared byincorporating the compound in the required amount in the appropriatesolvent with various other ingredients as required. Sterilizingprocedures, such as filtration, then follow. Typically, dispersions aremade by incorporating the compound into a sterile vehicle which alsocontains the dispersion medium and the required other ingredients asindicated above. In the case of a sterile powder, the preferred methodsinclude vacuum drying or freeze drying to which any required ingredientsare added.

Suitable pharmaceutical carriers include sterile'water; saline,dextrose; dextrose in water or saline; condensation products of castoroil and ethylene oxide combining about 30 to about 35 moles of ethyleneoxide per mole of castor oil, liquid acid, lower alkanols, oils such ascorn oil, peanut oil, sesame oil and the like, with emulsifiers such asmono- or di-glyceride of a fatty acid, or a phosphatide, e.g., lecithin,and the like, glycols, polyalkylene glycols, aqueous media in thepresence of a suspending agent, for example, sodiumcarboxymethylcellulose, sodium alginate, poly(vinylpyrrolidone), and thelike, alone, or with suitable dispensing agents such as lecithin,polyoxyethylene stearate, and the like. The carrier can also containadjuvants such as preserving stabilizing, wetting, emulsifying agentsand the like together with the penetration enhancer. In all cases thefinal form, as noted, must be sterile and must also be able to passreadily through an injection device such as a hollow needle. The properviscosity can be achieved and maintained by the proper choice ofsolvents or excipients. Moreover, the use of molecular or particulatecoatings such as lecithin, the proper selection of particle size indispersions, or the use of materials with surfactant properties may beutilized.

In accordance with the present invention, there are providedcompositions of camptothecin derivatives and methods useful for the invivo delivery of di-ester of 10-HCPT derivatives in the form ofnanoparticles, which are suitable for any route administrations.

U.S. Pat. Nos. 5,916,596, 6,506,405 and 6,537,579 teach the preparationof nanoparticles from the biocompatible polymers, such as albumin. Thus,in accordance with the present invention, there are provided methods forthe formation of nanoparticles of present invention by a solventevaporation technique from an oil-in-water emulsion prepared underconditions of high shear forces (e.g., sonication, high pressurehomogenization, or the like).

Thus, in accordance with the present invention, di-ester derivatives of10-HCPT are dissolved in a water miscible organic solvent (e.g., asolvent having greater than about 10% solubility in water, such as, forexample, ethanol) is added to the oil phase at a final concentration inthe range of about 1-99% (v/v), more preferably in the range of about5-25% (v/v) of the total organic phase. The water miscible organicsolvent can be selected from such solvents as ethyl acetate, ethanol,tetrahydrofuran, dioxane, acetonitrile, acetone, dimethyl sulfoxide,dimethyl formamide, methylpyrrolidinone, and the like. Alternatively,the mixture of water immiscible solvent with the water miscible solventis prepared first, followed by dissolution of the pharmaceuticallyactive agent in the mixture.

Next, a protein (e.g., human serum albumin) is added (into the aqueousphase) to act as a stabilizing agent for the formation of stablenanodroplets. Protein is added at a concentration in the range of about0.05 to 25% (w/v), more preferably in the range of about 0.5-5% (w/v).Unlike conventional methods for nanoparticle formation, no surfactant(e.g. sodium lauryl sulfate, lecithin, Tween® 80, Pluronic® F-68 and thelike) is added to the mixture. Optionally, a sufficient amount of thefirst organic solvent (e.g., chloroform) is dissolved in the aqueousphase to bring it close to the saturation concentration. A separate,measured amount of the organic phase (which now contains thepharmacologically active agent, the first organic solvent and the secondorganic solvent) is added to the saturated aqueous phase, so that thephase fraction of the organic phase is between about 0.5-15% (v/v), andmore preferably between 1% and 8% (v/v).

An emulsion is formed by homogenization under high pressure and highshear forces. Such homogenization is conveniently carried out in ahigh-pressure homogenizer, typically operated at pressures in the rangeof about 3,000 up to 30,000 psi. Preferably, such processes are carriedout at pressures in the range of about 6,000 up to 25,000 psi. Theresulting emulsion comprises very small nanodroplets of the nonaqueoussolvent (containing the dissolved pharmacologically active agent) andvery small nanodroplets of the protein-stabilizing agent. Acceptablemethods of homogenization include processes imparting high shear andcavitation such as high pressure homogenization, high shear mixers,sonication, high shear impellers, and the like.

Finally, the solvent is evaporated under reduced pressure to yield acolloidal system composed of protein-coated nanoparticles ofpharmacologically active di-ester of 10-HCPT and protein. Acceptablemethods of evaporation include the use of rotary evaporators, fallingfilm evaporators, spray driers, freeze driers, and the like. Thus, acolloidal dispersion system (pharmacologically active agent and protein)in the form of extremely small nanoparticles (i.e., particles in therange of about 10-200 nm diameter) can be sterile-filtered. Thepreferred size range of the particles is between about 50-170 nm,depending on the formulation and operational parameters.

Colloidal systems prepared in accordance with the present invention canbe further converted into powder form by removal of the water, e.g., bylyophilization at a suitable temperature-time profile. The protein(e.g., human serum albumin) itself acts as a cryoprotectant, and thepowder is easily reconstituted by addition of water, saline or buffer,without the need to use such conventional cryoprotectants as mannitol,sucrose, glycine, and the like. While not required, it is of courseunderstood that conventional cryoprotectants can be added to inventionformulations if so desired.

The polymeric shell containing solid or liquid cores ofpharmacologically active agent allows for the delivery of high doses ofthe pharmacologically active agent in relatively small volumes. Thisminimizes patient discomfort at receiving large volumes of fluid andminimizes hospital stay. In addition, the walls of the polymeric shellor coating are generally completely degradable in vivo by proteolyticenzymes (e.g., when the polymer is a protein), resulting in no sideeffects from the delivery system as is the case with currentformulations.

A number of biocompatible materials can be employed in the practice ofthe present invention for the formation of a polymeric shell. As usedherein, the term “biocompatible” describes a substance that does notappreciably alter or affect in any adverse way, the biological systeminto which it is introduced. Several biocompatible materials may beemployed in the practice of the present invention for the formation of apolymeric shell. For example, naturally occurring biocompatiblematerials such as proteins, polypeptides, oligopeptides,polynucleotides, polysaccharides (e.g., starch, cellulose, dextrans,alginates, chitosan, pectin, hyaluronic acid, and the like), lipids, andso on, are candidates for such modification.

As examples of suitable biocompatible materials, naturally occurring orsynthetic proteins may be employed. Examples of suitable proteinsinclude albumin, insulin, hemoglobin, lysozyme, immunoglobulins,α-2-macroglobulin, fibronectin, vitronectin, fibrinogen, casein and thelike, as well as combinations of any two or more thereof. Similarly,synthetic polymers are also good candidates for preparation of the drugformulation. Examples include polyalkylene glycols (e.g., linear orbranched chain), polyvinyl alcohol, polyacrylates, polyhydroxyethylmethacrylate, polyacrylic acid, polyethyloxazoline, polyacrylamides,polyisopropyl acrylamides, polyvinyl pyrrolidinone,polylactide/glycolide and the like, and combinations thereof, are goodcandidates for the biocompatible polymer in the invention formulation.

These biocompatible materials can also be employed in several physicalforms such as gels, crosslinked or uncrosslinked to provide matricesfrom which the pharmacologically active ingredient, for examplepaclitaxel, can be released by diffusion and/or degradation of thematrix. Temperature sensitive materials can also be utilized as thedispersing matrix for the invention formulation. Thus for example, thecamptothecin di-ester may be injected in a liquid formulation of thetemperature sensitive material (e.g., copolymers of polyacrylamides orcopolymers of polyalkylene glycols and polylactide/glycolides) which gelat the tumor site and provide slow release of di-ester of 10-RCPT.

Particles of biologic substantially completely contained within apolymeric shell, or associated therewith, prepared as described herein,are delivered neat, or optionally as a suspension in a biocompatiblemedium. This medium may be selected from water, buffered aqueous media,saline, buffered saline, optionally buffered solutions of amino acids,optionally buffered solutions of proteins, optionally buffered solutionsof sugars, optionally buffered solutions of carbohydrates, optionallybuffered solutions of vitamins, optionally buffered solutions ofsynthetic polymers, lipid-containing emulsions, and the like.

In addition, the polymeric shell optionally can be modified by asuitable agent, wherein the agent is associated with the polymeric shellthrough an optional covalent bond. Covalent bonds contemplated for suchlinkages include ester, ether, urethane, di-ester, amide, secondary ortertiary amine, phosphate ester, sulfate ester, and the like bonds.Suitable agents contemplated for this optional modification of thepolymeric shell include synthetic polymers (polyalkylene glycols (e.g.,linear or branched chain polyethylene glycol), polyvinyl alcohol,polyhydroxyethyl methacrylate, polyacrylic acid, polyethyloxazoline,polyacrylamide, polyvinyl pyrrolidinone, and the like), phospholipids(such as phosphatidyl choline (PC), phosphatidyl ethanolamine (PE),phosphatidyl inositol (PI), sphingomyelin, and the like), proteins (suchas enzymes, antibodies, and the like), polysaccharides (such as starch,cellulose, dextrans, alginates, chitosan, pectin, hyaluronic acid, andthe like), chemical modifying agents (such as pyridoxal 5′-phosphate,derivatives of pyridoxal, dialdehydes, diaspirin esters, and the like),or combinations of any two or more thereof.

The prepared nanoparticle with this invention can be administered by anyacceptable route including, but not limited to, orally, intramuscularly,transdermally, intravenously, through an inhaler or other air bornedelivery systems, and the like. When preparing the composition forinjection, particularly for intravenous delivery, the continuous phasepreferably comprises an aqueous solution of tonicity modifiers, bufferedto a pH below 7, more preferably below 6.

The prepared nanoparticles of this invention can be enclosed in a hardor soft capsule, can be compressed into tablets, or can be incorporatedwith beverages, food or otherwise into the diet. Capsules can beformulated by mixing the nanoparticle with a pharmaceutical diluentwhich is inert and inserting this mixture into a hard gelatin capsulehaving the appropriate size. If soft capsules are desired, a slurry ofthe compound with an acceptable vegetable, light petroleum, or otherinert oil can be encapsulated by machine into a gelatin capsule. Thepercentage of the final composition and the preparations may, of course,be varied and may conveniently range between 1 and 90% of the weight ofthe final form, e.g., tablet. The amount in such therapeutically usefulcompositions is such that a suitable dosage will be obtained. Preferredcompositions according to the current invention are prepared so that anoral dosage unit form contains between about 5 to about 50% by weight (%w) in dosage units weighing between 50 and 1000 mg.

In addition, the compounds and the formulations of the present inventioncan be used in combination with other drugs and formulations for thetreatment of cancers such as Taxol®, Taxotere®, or their derivatives,VP-16, 5-FU, as well as cisplatin and derivatives thereof.

Another important feature of the method provided by the presentinvention relates to the relatively low apparent overall toxicity of the10-HCPT derivatives administered in accordance with the teachingsherein. Overall toxicity can be judged using various criteria. Forexample, loss of body weight in a subject over 10% of the initiallyrecorded body weight (i.e., before treatment) can be considered as onesign of toxicity. In addition, loss of overall mobility and activity andsigns of diarrhea or cystitis in a subject can also be interpreted asevidence of toxicity.

Other features of the present invention will become apparent in view ofthe following examples, which are given for illustration of theinvention and are not intended to be limiting thereof.

Example 1

This example illustrates the preparation of camptothecin10,20-di-O-butyrate (CY1). To a round-bottomed flask was added10-hydroxycamptothecin (3.0 g, 8.24 mmol), butyric anhydride (60 mL),followed by adding concentrated sulfuric acid (10 drops) dropwise atroom temperature. The obtained mixture was stirred at about 100° C. forovernight. After cooling to room temperature, the mixture was pouredinto 350 mL petroleum ether portion by portion while stirring. Afterstirring for about 1 h, the crude product precipitated was collected byfiltration. The crude product was then dissolved into DCM/H₂O. Theorganic layer was washed with 5% NaHCO₃, brine, dried (MgSO₄) andconcentrated. The residue was purified by flash silica gelchromatography eluted with THF/DCM (3-5%) to afford the white solid (3.7g, 89%). Anal. Calcd for (C₂₈H₂₈N₂O₇+H)⁺ and (C₂₈H₂₈N₂O₇+Na)⁺: 505 and527. Found: 505 and 527.

Example 2

This example illustrates the preparation of camptothecin10,20-di-O-isobutyrate (CY2). To a round-bottomed flask was added10-hydroxycamptothecin (1.2 g, 3.29 mmol), isobutyric anhydride (40 mL),and followed by adding concentrated sulfuric acid (8 drops) dropwise atroom temperature. The reaction mixture was stirred at 100° C. for 16 h.After cooling to room temperature, the mixture was poured into 250 mLpetroleum ether portion by portion while stirring. After stirring forabout 45 min, the crude product precipitated was collected by vacuumfiltration. The crude product was partitioned with DCM (200 mL) and 5%NaHCO₃ (80 mL), brine. The organic layer was washed with brine (1×100mL) and dried over anhydrous MgSO₄, filtered and concentrated in vacuo.The residue was purified by flash silica gel column chromatography (THFin DCM, 3-5%) to afford a white solid (1.59 g, 96%). Anal. Calcd for(C₂₈H₂₈N₂O₇+H)⁺ and (C₂₈H₂₈N₂O₇+Na)⁺: 505 and 527. Found: 505 and 527.

Example 3

This example illustrates the preparation of camptothecin10,20-di-O-hexonate (CY4). To a round-bottomed flask was added10-hydroxycamptothecin (1.8 g, 4.94 mmol), hexanoic anhydride (50 mL),and followed by adding concentrated sulfuric acid (9 drops) dropwise atroom temperature. The reaction mixture was stirred at 100° C. for 17 h.After cooling to room temperature, the mixture was poured into 300 mLpetroleum ether portion by portion while stirring. After stirring forabout 1 h, the crude product precipitated was collected by vacuumfiltration. The crude product was partitioned with DCM (250 mL) and 5%NaHCO₃ (80 mL), brine. The organic layer was washed with brine (1×100mL) and dried over anhydrous MgSO₄, filtered and concentrated in vacuo.The residue was purified by flash silica gel column chromatography (THFin DCM, 5-10%) to afford a white solid (2.38 g, 86%). Anal. Calcd for(C₃₂H₃₆N₂O₇+H)⁺ and (C₃₂H₃₆N₂O₇+Na)⁺: 561 and 583. Found: 561 and 583.

Example 4

This example illustrates the preparation of camptothecin10,20-di-O-tert-butylaminoacetate (CY28). To a three neck flask wasadded 10-hydroxycamptothecin (1.8 g, 4.89 mmol), BOC-glycine (3.9 g, 22mmol) and DMF (40 mL), at 0° C. DMAP (548 mg, 4.89 mmol) and DCC (5.0 g,24.45 mmol) were added sequentially to the reaction mixture. After theaddition, the reaction mixture was stirred at room temperature for 72 h.Cooled hexane (150 mL) was added to the brownish mixture while stirring.After one hour, the mixture was filtered. The residue was coevaporatedwith toluene once and purified by flash silica gel column chromatography(THF:DCM, 1:10) to afford a yellow solid (238.1 mg, 7.2%). ¹H NMR(CDCl₃, 500 MHz): δ 8.34 (s, 1H), 8.25 (d, J=7.4 Hz, 1H), 8.01 (s, 1H),7.71 (d, J=2.1 Hz, 1H), 7.57 (dd, J=7.4, 2.0 Hz, 1H), 5.54 (AB, Δy=116.5Hz, J=13.8 Hz, 2H), 5.27 (d, J=1.7 Hz, 2H), 5.25 (t, J=4.7 Hz, 1H), 4.99(t, J=5.2 Hz, 1H), 4.79 (s, 2H), 4.54 (d, J=4.3 Hz, 2H), 2.28-2.13 (m,2H), 1.58 (s, 9H), 1.46 (s, 9H), 0.99 (t, J=5.9 Hz, 3H). ESI-MS: calcd.for C₃₄H₃₉N₄O₁₁Na (M+Na+H)⁺: 702. Found: 702.

Example 5

This example illustrates the preparation of camptothecin10,20-di-O-methoxyacetate (CY30). To a three neck flask was added10-hydroxycamptothecin (424 mg, 1.16 mmol), methoxyacetic acid (356 μL,4.64 mmol) and DMF (40 mL), at 0° C. DMAP (130 mg, 1.16 mmol) and DCC(1.2 g, 5.8 mmol) were added sequentially to the reaction mixture. Afterthe addition, the reaction mixture was stirred at room temperature for48 h. Cooled hexane (100 mL) was added to the brownish mixture whilestirring. After one hour, the mixture was filtered. The residue wascoevaporated with toluene once and purified by flash silica gel columnchromatography (THF:DCM, 1:7) to afford a yellow solid. ¹H NMR (CDCl₃,500 MHz): δ 8.34 (s, 1H), 8.23 (d, J=7.2 Hz, 1H), 7.76 (d, J=2.2 Hz,1H), 7.61 (dd, J=7.2, 2.0 Hz, 1H), 7.20 (s, 1H), 5.58 (AB, Δy=120 Hz,J=14.2 Hz, 2H), 5.30 (s, 2H), 4.39 (s, 2H), 4.23 (dd, J=23.3, 13.5 Hz,2H), 3.59 (s, 3H), 3.46 (s, 3H), 2.28-2.13 (m, 2H), 0.99 (t, J=6.0 Hz,3H). ESI-MS: calcd. for C₂₆H₂₄N₂O₉Na (M+Na)⁺: 531. Found: 531.

Example 6

This example illustrates the preparation of camptothecin10,20-di-O-aminoacetate (CY32). To a solution of camptothecin10,20-di-O-tert-butylaminoacetate (40 mg, 0.06 mmol) in MeOH (3 mL) at0° C. was added saturated solution of HCl in dioxane (0.5 mL) within 5min. After addition, the reaction mixture was warmed up to rt and stoodfor 2 h. The reaction mixture was stored at 4° C. overnight. TLC showedthe complete disappearance of starting material. The reaction mixturewas washed with chloroform (3×5 mL) and subjected to lyophilization toafford the desired product as a yellow foam (12.7 mg, 38.5%). ¹H NMR(CDCl₃, 500 MHz): δ 7.94 (s, 1H), 7.76 (d, J=9.2 Hz, 1H), 7.25 (dd,J=9.5, 2.6 Hz, 1H), 7.18 (s, 1H), 6.92 (d, J=2,5 Hz, 1H), 5.57 (AB,Δy=86 Hz, J=16.3 Hz, 2H), 4.35 (d, J=4.8 Hz, 2H), 4.01 (s, 2H), 3.88 (s,2H), 2.36-2.26 (m, 2H), 1.10 (t, J=7.3 Hz, 3H).

Example 7

This example illustrates the preparation of Camptothecin10,20-di-O-benzoate (CY55). To a solution of 10-hydroxy camptothecin(605 mg, 1.66 mmol) in pyridine (50 mL) at 0° C. was added benzoicanhydride (2.25 g, 996 mmol). The reaction mixture was stirred at roomtemperature for 24 h and concentrated on rotavapor to remove most of thepyridine. Cold hexanes (100 mL) was added to the residue. The suspensionwas stored at 0° C. for 2 days and filtered. The solid was collected andpurified by flash silica gel column chromatography (THF:DCM, 1:20) toafford a yellow solid (647 mg, 68.1%). ¹H NMR (CDCl₃, 500 MHz): δ 8.36(s, 1H), 8.25-8.23 (m, 2H), 8.20 (d, J=7.4 Hz, 1H), 7.81 (d, J=2.5 Hz,1H), 7.70-7.61 (m, 3H), 7.56-7.48 (m, 4H), 5.77 (d, J=17.2 Hz, 1H), 5.47(d, J=17.1 Hz, 1H), 5.34-5.25 (m, 2H), 2.48-2.43 (m, 1H), 2.36-2.27 (m,1H), 1.68 (brs, 1H), 1.10 (t, J=7.5 Hz, 3H). ESI-MS: calcd. forC₃₄H₂₅N₂O₇ (M+H)⁺: 573. Found: 573.

Example 8

This example illustrates the preparation of 7-ethylcamptothecin10,20-di-O-benzoate (CY57). To a round-bottomed flask was added SN-38(480 mg, 1.22 mmol), propionic anhydride (50 mL), followed by addingconcentrated sulfuric acid (14 drops) dropwise at room temperature. Theobtained mixture was stirred at about 100° C. for 17 h. After cooling toroom temperature, the mixture was poured into 350 mL petroleum etherportion by portion while stirring. After stirring for about 1 hr andstanding at 0° C. for 48 h, the crude product was collected byfiltration. The crude product was then dissolved into DCM/H₂O. Theorganic layer was washed with 5% NaHCO₃, brine, dried (MgSO₄) andconcentrated. The residue was purified by flash silica gelchromatography eluted with THF/DCM (2-5%) to afford the white solid (268mg, 44%). ¹H NMR (CDCl₃, 500 MHz): δ ESI-MS: calcd. forC₂₈H₂₉N₂O₇(M+H)⁺: 505. Found: 505.

Example 9

This example illustrates the preparation of 7-ethylcamptothecin10,20-di-O-hexanoate (CY189). To a solution of SN38 (1.62 g, 4.1 mmol)in pyridine (125 mL) at 0° C. was added hexanoic anhydride (5.7 mL, 24.2mmol). The reaction mixture was stirred at room temperature for 21 h andquenched by 20 mL of methanol. The mixture was stirred for additional 2h at room temperature and concentrated on rotavapor to dryness. Theresidue was purified by flash silica gel column chromatography (THF:DCM,1:30) to afford a pale yellow solid (2.33 g, 96%). ¹H NMR (CDCl₃, 500MHz): δ 8.20 (d, J=9.2 Hz, 1H), 7.84 (s, 1H), 7.56 (d, J=9.2 Hz, 1H),7.17 (s, 1H), 5.68 (d, J=17.1 Hz, 1H), 5.41 (d, J=17.1 Hz, 1H), 5.24 (d,J=3.8 Hz, 2H), 3.15 (dd, J=15.2, 7.8 Hz, 2H), 2.66 (t, J=7.5 Hz, 2H),2.35 (t, J=7.5 Hz, 4H, methylene), 1.66-1.25 (m, 15H), 0.97 (t, J=6.8Hz, 3H), 0.89-0.85 (m, 6H). ESI-MS: calcd. for C₃₄H₄₁N₂O₇ (M+H)⁺: 589.Found: 589.

Example 10

This example illustrates the preparation of 7-ethylcamptothecin10,20-di-O-linoleicate (CY201). To a solution of SN38 (279 mg, 0.71mmol) in pyridine (30 mL) at 0° C. was added linoleic anhydride (2 mL,4.24 mmol). The reaction mixture was stirred at room temperature for 21h and quenched by 20 mL of methanol. The mixture was stirred foradditional 2 h at room temperature and concentrated on rotavapor todryness. The residue was purified by flash silica gel columnchromatography (THF:DCM, 1:45) to afford a pale yellow solid (283 mg,43%). ¹H NMR (CDCl₃, 500 MHz): δ 8.29 (d, J=9.2 Hz, 1H), 7.85 (d, J=2.4Hz, 1H), 7.58 (dd, J=9.2, 2.4 Hz, 1H), 7.31 (s, 1H), 5.68 (d, J=17.2 Hz,1H), 5.41-5.21 (m, 1H), 3.16 (dd, J=15.2, 7.6 Hz, 2H), 2.78-2.73 (m,6H), 2.66 (t, J=7.5 Hz, 2H), 2.55-2.46 (m, 2H), 2.35 (t, J=7.5 Hz, 4H),1.99-1.85 (m, 4H), 1.84-1.78 (m, 4H), 1.66-1.25 (m, 31H), 0.97 (t, J=6.9Hz, 3H), 0.89-0.85 (m, 6H). ESI-MS: calcd. for C₅₈H₈₁N₂O₇ (M+H)⁺: 918.Found: 918.

Example 11

This example illustrates the preparation of 7-ethylcamptothecin10,20-di-O-decanoicate (CY203). To a solution of SN38 (203 mg, 0.51mmol) in pyridine (25 mL) at 0° C. was added decanoic anhydride (1.39 g,4.24 mmol). The reaction mixture was stirred at room temperature for 21h and quenched by 10 mL of methanol. The mixture was stirred foradditional 3 h at room temperature and concentrated on rotavapor todryness. The residue was purified by flash silica gel columnchromatography (THF:DCM, 1:45) to afford a pale yellow solid (292 mg,81%). ¹H NMR (CDCl₃, 500 MHz): δ 8.22 (d, J=9.1 Hz, 1H, 12-H), 7.82 (d,J=2.5 Hz, 1H, 9-H), 7.56 (dd, J=9.2, 2.4 Hz, 1H, 11-H), 7.19 (s, 1H,14-H), 5.68 (d, J=17.1 Hz, 1H, 17-H), 5.41 (d, J=17.2 Hz, 1H, 17-H),5.24 (d, J=3.8 Hz, 2H, 5-H), 3.15 (dd, J=15.4, 7.7 Hz, 2H,C7-methylene), 2.66 (t, J=7.5 Hz, 2H, methylene), 2.35 (t, J=7.5 Hz, 4H,methylene), 1.66-1.25 (m, 31H), 0.97 (t, J=6.8 Hz, 3H, methyl),0.89-0.85 (m, 6H, methyl). ESI-MS: calcd. for C₄₂H₅₇N₂O₇ (M+H)⁺: 701.Found: 701.

Example 12

This example illustrates the preparation of 7-ethylcamptothecin10,20-di-O-myristate (CY204). To a solution of SN38 (332 mg, 0.85 mmol)in pyridine (25 mL) at 0° C. was added decanoic anhydride (2.24 g, 5.11mmol). The reaction mixture was stirred at room temperature for 26 h andquenched by 10 mL of methanol. The mixture was stirred for additional 3h at room temperature and concentrated on rotavapor to dryness. Theresidue was purified by flash silica gel column chromatography (THF:DCM,1:45) to afford a white solid (539 mg, 78%). ¹H NMR (CDCl₃, 500 MHz): δ8.22 (d, J=9.2 Hz, 1H), 7.82 (d, J=2.4 Hz, 1H), 7.56 (dd, J=9.1, 2.4 Hz,1H), 7.20 (s, 1H), 5.67 (d, J=17.2 Hz, 1H), 5.41 (d, J=17.2 Hz, 1H),5.25 (d, J=4.0 Hz, 2H), 3.15 (dd, J=15.2, 7.8 Hz, 2H), 2.66 (t, J=7.8Hz, 2H), 2.33 (t, J=7.5 Hz, 4H), 1.66-1.25 (m, 47H), 0.97 (t, J=6.8 Hz,3H), 0.89-0.80 (m, 6H). ESI-MS: calcd. for C₅₀H₇₃N₂O₇ (M+H)⁺: 814.Found: 814.

Example 13

This example illustrates the preparation of 7-ethylcamptothecin10,20-di-O-laurate (CY205). To a solution of SN38 (245 mg, 0.63 mmol) inpyridine (30 mL) at 0° C. was added lauric anhydride (1.44 g, 3.76mmol). The reaction mixture was stirred at room temperature for 24 h andquenched by 10 mL of methanol. The mixture was stirred for additional 3h at room temperature and concentrated on rotavapor to dryness. Theresidue was purified by flash silica gel column chromatography (THF:DCM,1:40) to afford a white solid (388 mg, 82%). ¹H NMR (CDCl₃, 500 MHz): δ8.22 (d, J=9.1 Hz, 1H), 7.83 (d, J=2.1 Hz, 1H), 7.56 (dd, J=9.1, 2.1 Hz,1H), 7.19 (s, 1H), 5.68 (d, J=17.2 Hz, 1H), 5.42 (d, J=17.2 Hz, 1H),5.25 (d, J=4.0 Hz, 2H), 3.15 (dd, j=15.2, 7.8 Hz, 2H), 2.66 (t, J=7.8Hz, 2H), 2.52-2.48 (m, 2H), 2.33 (t, J=7.5 Hz, 4H), 1.66-1.25 (m, 37H),0.97 (t, J=6.8 Hz, 3H), 0.89-0.80 (m, 6H). ESI-MS: calcd. for C₄₆H₆₅N₂O₇(M+H)⁺: 757. Found: 757.

Example 14

This example illustrates the preparation of 7-ethylcamptothecin10,20-di-O-stearate (CY206). To a solution of SN38 (195 mg, 0.50 mmol)in pyridine (40 mL) at 0° C. was added stearic anhydride (1.64 g, 2.98mmol). The reaction mixture was stirred at room temperature for 48 h andquenched by 10 mL of methanol. The mixture was stirred for additional 5h at room temperature and concentrated on rotavapor to dryness. Theresidue was purified by flash silica gel column chromatography (THF:DCM,1:40) to afford a yellow solid (396 mg, 86%). ¹H NMR (CDCl₃, 500 MHz): δ8.21 (d, J=9.1 Hz, 1H), 7.82 (d, J=2.2 Hz, 1H), 7.55 (dd, J=9.1, 2.2 Hz,1H), 7.19 (s, 1H), 5.68 (d, J=17.3 Hz, 1H), 5.41 (d, J=17.3 Hz, 1H),5.24 (d, J=4.0 Hz, 2H), 3.15 (dd, J=15.2, 7.6 Hz, 2H), 2.66 (t, J=7.6Hz, 2H), 2.51-2.42 (m, 2H), 2.36 (t, J=7.8 Hz, 4H), 1.68-1.25 (m, 61H),0.97 (t, J=6.8 Hz, 3H), 0.87-0.80 (m, 6H). ESI-MS: calcd. for C₅₈H₈₉N₂O₇(M+H)⁺: 926. Found: 926.

Example 15

This example illustrates the preparation of 7-ethylcamptothecin10,20-di-O-(2′-methylheptanoate) (CY212). To a solution of SN38 (300 mg,0.76 mmol) in pyridine (40 mL) at 0° C. was added 2′-methylheptanoicanhydride (1.24 g, 4.59 mmol). The reaction mixture was stirred at roomtemperature for 16 h and quenched by 10 mL of methanol. The mixture wasstirred for additional 4 h at room temperature and concentrated onrotavapor to dryness. The residue was purified by flash silica gelcolumn chromatography (THF:DCM, 1:30) to afford a yellow solid (400 mg,81%). ¹H NMR (CDCl₃, 500 MHz): δ 8.20 (dd, J=9.0, 4.1 Hz, 1H), 7.78 (d,J=2.2 Hz, 1H), 7.54 (dd, J=9.1, 2.2 Hz, 1H), 7.20 (d, J=0.8 Hz, 1H),5.67 (dd, J=17.3, 2.1 Hz, 1H), 5.41 (dd, J=17.3, 2.1 Hz, 1H), 5.25 (d,J=1.7 Hz, 2H), 3.15 (dd, J=15.0, 7.6 Hz, 2H), 2.64-2.59 (m, 2H),2.47-2.38 (m, 2H), 2.35-2.25 (m, 2H), 1.75-1.25 (m, 23H), 1.08 (t, J=7.4Hz, 3H), 1.02-0.94 (m, 6H). ESI-MS: calcd. for C₃₈H₄₉N₂O₇ (M+H)⁺: 645.Found: 645.

Example 16

This example illustrates the preparation of 7-ethylcamptothecin10,20-di-O-palmitate (CY213). To a solution of SN38 (246 mg, 0.63 mmol)in pyridine (40 mL) at 0° C. was added palmitic anhydride (1.87 g, 3.78mmol). The reaction mixture was stirred at room temperature for 24 h andquenched by 10 mL of methanol. The mixture was stirred for additional 4h at room temperature and concentrated on rotavapor to dryness. Theresidue was purified by flash silica gel column chromatography (THF:DCM,1:30) to afford a yellow solid (500 mg, 92%). ¹H NMR (CDCl₃, 500 MHz): δ8.23 (dd, J=9.2, 4.0 Hz, 1H), 7.82 (d, J=2.2 Hz, 1H), 7.56 (dd, J=9.2,2.2 Hz, 1H), 7.21 (s, 1H), 5.68 (dd, J=17.2, 2.2 Hz, 1H), 5.42 (dd,J=17.2, 2.1 Hz, 1H), 5.25 (d, J=1.8 Hz, 2H), 3.15 (dd, J=15.0, 7.8 Hz,2H), 2.66 (t, J=7.5 Hz, 2H), 2.52-2.43 (m, 2H), 2.35 (t, J=7.5 Hz, 3H),1.85-1.25 (m, 54H), 1.08 (t, J=7.2 Hz, 3H), 0.96-0.86 (m, 6H), ESI-MS:calcd. for C₅₄H₈₁N₂O₇ (M+H)⁺: 870.

Found: 870.

Example 17

This example showed the in vitro growth inhibition experiments for thecompounds in the invention on MX-1 (human breast carcinoma) cells. Thecytotoxicity assay was quantitated using the Promega CellTiter Blue CellViability Assay. Briefly, cells (5000 cells/well) were plated onto96-well microtiter plates in RPMI 1640 medium supplemented with 10% FBSand incubated at 378 C in a humidified 5% CO₂ atmosphere. After 24 h,cells were exposed to various concentrations of compound in DMSO andcultured for another 72 h. 100 μl of media were removed and 20 μl ofPromega CellTiter Blue reagent were added to each well and shaken tomix. After 4 hours of incubation at 37° C. in a humidified 5% CO₂atmosphere, the plates were read at 544ex/620em. The fluorescenceproduced is proportional to the number of viable cells. After plottingfluorescence produced against drug concentration, the IC₅₀ wascalculated as the half-life of the resulting non-linear regression. Thedata showed in Table 1.

TABLE 1 IC₅₀ of camptothecin analogs.

Substitutes IC₅₀ ID R₄ R MW μM CY1 H COCH₂CH₂CH₃ 504.53 151 CY2 HCOCH(CH₃)₂ 504.53   22.8 CY4 H COCH₂CH₂CH₂CH₂CH₃ 560.64    7.8 CY28 HCOCH₂NHCOOtBu 678.69 221 CY30 H COCH₂OCH₃ 508.48 372 CY32 H COCH₂NH₂478.45 242 CY55 H COPh 572.56 739 CY57 Et COCH₂CH₃ 504.54  27 CY189 EtCO(CH₂)₄CH₃ 588.69    3.4 CY201 Et CO(CH₂)₈CH═CHCH═CH(CH₂)₄CH₃ 917.26500 CY203 Et CO(CH₂)₈CH₃ 700.90 2000  CY204 Et CO(CH₂)₁₂CH₃ 813.12  >50*CY205 Bt CO(CH₂)₁₀CH₃ 757.01  >50* CY206 Et CO(CH₂)₁₆CH₃ 925.33  >50*CY212 Et CO(CH₂)₃CH(CH₃)CH₂CH₃ 644.80 294 CY213 Et CO(CH₂)₁₄CH₃ 869.22 >50* 10-HCPT H H 348.35   13.5 SN-38 Et H 392.40 267 Note: IC₅₀ > 50*means the compounds insoluble at higher concentration.

Example 18

This example illustrates the preparation of camptothecin10,20-di-O-hexonate-albumin compositions. 30 mg camptothecin10,20-di-O-hexonate (as prepared in Example 3) was dissolved in 3.0 mLmethylene chloride/methanol (9/1). The solution was then added into 27.0mL of human serum albumin solution (3% w/v). The mixture was homogenizedfor 5 minutes at low RPM (Vitris homogenizer model: Tempest I.Q.) inorder to form a crude emulsion, and then transferred into ahigh-pressure homogenizer (Avestin). The emulsification was performed at9000-40,000 psi while recycling the emulsion for at least 5 cycles. Theresulting system was transferred into a Rotavap and solvent was rapidlyremoved at 40° C., at reduced pressure (30 mm Hg), for 20-30 minutes.The resulting dispersion was translucent and the typical averagediameter of the resulting particles was in the range 50-220 nm(Z-average, Malvern Zetasizer). The dispersion was further lyophilizedfor 48 hours. The resulting cake could be easily reconstituted to theoriginal dispersion by addition of sterile water or saline. The particlesize after reconstitution was the same as before lyophilization. Itshould be recognized that the amounts, types and proportions of drug,solvents, proteins used in this example are not limiting in anyway.

Example 19

This example illustrates the formation of nanoparticles of camptothecindi-ester by using cavitation and high shear forces during a sonicationprocess. Thus, 20 mg Camptothecin 10,20-di-O-hexonate (as prepared inExample 3) was dissolved in 1.0 mL methylene chloride. The solution wasadded to 4.0 mL of human serum abumin solution (5% w/v). The mixture washomogenized for 5 minutes at low RPM (Vitris homogenizer, model: TempestI.Q.) in order to form a crude emulsion, and then transferred into a 40kHz sonicator cell. The sonicator was performed at 60-90% power at 0degrees for 1 min (550 Sonic Dismembrator). The mixture was transferredinto a Rotary evaporator, and methylene chloride is rapidly removed at40° C., at reduced pressure (30 mm Hg), for 20-30 minutes. The typicaldiameter of the resulting paclitaxel particles was 350-420 nm(Z-average, Malvern Zetasizer).

The dispersion was further lyophilized for 48 h without adding anycryoprotectant. The resulting cake could be easily reconstituted to theoriginal dispersion by addition of sterile water or saline. The particlesize after reconstitution was the same as before lyophilization.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1.-54. (canceled)
 55. A di-ester compound having the followingstructure:

wherein R₁, R₂, R₃, and R₄, which can be the same or different, arehydrogen, halogen, C₁-C₂₀ alkyl, C₁-C₈ alkoxyl, C₄-C₂₀ aryl or C₁-C₂₀silyl, R, which can be the same or different, is C₂-C₃₀ alkyl, C₂-C₂₂alkenyl, C₄-C₃₀ aryl, (CH₂)_(n)OR₅, (CH₂)_(n)SR₅, (CH₂)_(n)NR₅R₆ or(CH₂)_(n)COR₇, R₅ and R₆, which can be the same or different, are C₁-C₈alkyl or C₂-C₆ alkenyl, R₇ is hydroxy, C₁-C₂₀ alkyl, C₁-C₆ alkenyl,C₁-C₆ alkoxy, C₄-C₂₀ aryl, or NR₈R₉, R₈ and R₉, which can be the same ordifferent, are C₁-C₆ alkyl, and n is an integer of 1 to 8, or apharmaceutically acceptable salt thereof.
 56. The di-ester compound ofclaim 55, wherein each of R₁, R₂, R₃ and R₄ is H, and R is (CH₂)_(n)SR₅,R₅ is C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₄-C₁₀ aryl, and n is 1 or
 2. 57.The di-ester compound of claim 55, wherein each of R₁, R₂ and R₃ is H,R₄ is CH₂CH₃, and R is (CH₂)_(n)SR₅, R₅ is C₁-C₆ alkyl, C₂-C₆ alkenyl,or C₄-C₁₀ aryl, and n is 1 or
 2. 58. The di-ester compound of claim 55,wherein each of R₁, R₂ and R₃ is H, R₄ is Si(CH₃)₂C(CH₃)₃, and R is(CH₂)_(n)SR₅, R₅ is C₁-C₆ alkyl, C₂-C₆ alkenyl or C₄-C₁₀ aryl, and n is1 or
 2. 59. The di-ester compound of claim 55, wherein R₁ is CH₂N(CH₃)₂,each of R₂, R₃ and R₄ is H, and R is (CH₂)_(n)SR₅, R₅ is C₁-C₆ alkyl,C₂-C₆ alkenyl or C₄-C₁₀ aryl, and n is 1 or
 2. 60. A method to inhibitthe enzyme topoisomerase I in an animal in need thereof comprisingadministering to the animal an effective amount of a compositioncomprising at least one di-ester compound of claim
 55. 61. A method totreat cancer in a patient comprising administering a compositioncomprising at least one di-ester compound of claim 55 to said patient inan amount effective to treat said cancer.
 62. The method of claim 61,wherein said cancer is lung, breast, colon, prostate, melanoma,pancreas, stomach, liver, brain, kidney, uterus, cervix, ovaries,urinary tract, gastrointestinal, or leukemia.
 63. The method of claim61, wherein said cancer is solid tumor or blood borne tumor.
 64. Themethod of claim 61, wherein said composition is administered orally,parenterally, intramuscularly, transdermally or by an airborne deliverysystem.
 65. The method of claim 61, wherein said composition is ananoparticle containing said at least one di-ester compound.
 66. Amethod to treat breast cancer in a patient comprising administering acomposition comprising at least one di-ester compound of claim 55 tosaid patient in an amount effective to treat said cancer.